Abstract

Secretory vesicles isolated from adrenal medulla were found to fuse in vitro in response to incubation with Ca2+. Intervesicular fusion was detected by electron microscopy and was indicated by the appearance of twinned vesicles in freeze-fractured suspensions of vesicles and in thin-sectioned pellet. Two types of fusion could be distinguished: Type I, occurring between 10−7 M and 10−4 M Ca2+, was specific for Ca2+, was inhibited by other divalent cations and was abolished by pretreatment of vesicles with glutaraldehyde, neuraminidase or trypsin. Fusion type I was linear with temperature. A second type of intervesicular fusion was elicited by Ca2+ in concentrations higher than 2.5 mM and was morphologically characterized by multiple fusions of secretory vesicles. This type of fusion was found to be similar to fusion of liposomes prepared from the membrane lipids of adrenal medullary secretory vesicles: Ca2+ could be replaced by other divalent cations, the effect of different divalent cations was additive and pretreatments attacking membrane proteins were ineffective. Fusion type II of intact secretory vesicles as well as liposome fusion was discontinuous with temperature. Liposome fusion could be detected within 35 ms and persisted for 180 min. Using liposomes containing defined Ca2+ concentrations we have not found a major influence of Ca2+ asymmetry on fusion. Incorporation of the ganglioside GM3, which is present in the membranes of intact adrenal medullary secretory vesicles did not change the properties of liposomes fusion. Using a Ca2+-selective electrode we have identified in secretory vesicle membranes both high affinity binding sites for Ca2+ (Kd = 1.6 · 10−6M) and low affinity sites (Kd = 1.2 · 10−4M).